Assay for JC virus antibodies

ABSTRACT

The disclosure relates to methods and reagents for analyzing samples for the presence of JC virus antibodies. Disclosed is a method that includes obtaining a biological sample from a subject (e.g., plasma, serum, blood, urine, or cerebrospinal fluid), contacting the sample with highly purified viral-like particles (HPVLPs) under conditions suitable for binding of a JCV antibody in the sample to an HPVLP, and detecting the level of JCV antibody binding in the sample to HPVLP. In one embodiment, determining the level of anti-JCV antibodies in the subject sample provides a method of identifying PML risk in a subject.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/294,048, filed Jan. 11, 2010, and U.S. Provisional Application No.61/316,193, Mar. 22, 2010, both of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods and reagents for analyzing samples forthe presence of JC virus antibodies.

BACKGROUND

Progressive Multifocal Leukoencephatopathy (PML) is an opportunisticinfection of the central nervous system (CNS) that associated withexposure to the JC virus (JCV), a polyoma virus that is be to bepathogenic in humans only under conditions of persistent immunesuppression or immune modulation. While the presence of JCV is requiredfor development of PML, PML risk is considered, in a not well-understoodway, to be associated with the convergence of multiple viral andhost-related factors that cause the virus to become pathogenic (Major,“Progressive Multifocal Leukoencephalopathy in Patients onImmunomodulatory Therapies” Annu. Rev. Med 61:35-47 (2010) [2009 Aug.31, Epub ahead of print]). Published studies reporting the prevalence ofJCV infection in the human population are varied. This information isbased on various types of studies including PCR analysis for viral DNAand detection of antibodies to JCV. Despite the prevalence of JCV in thepopulation, infection with JCV rarely results in PML, even inindividuals with documented immunosuppression.

Published reports on JCV DNA detection suggest the method to beinsensitive and of limited use for assessing exposure to JCV because JCVDNA has been rarely and inconsistently detected in the plasma, serum orperipheral blood mononuclear cells of JCV-infected PML patients.Detection of anti-JCV antibodies appears to be a more sensitive markerof JCV infection; however the reported results are variable. In 1973,Padgett and Walker published a study reporting a JCV seroprevalence of65-84% using a haemagglutination inhibition (HI) assay (Padgett andWalker, “Prevalence of antibodies in human sera agains JC virus, anisolate from a case of progressive multifocal leukoencephalopathy” J.Infect. Dis. 127:467-70, 1974. Later reports of JCV seroprevalence ratesusing the HI assay or ELISA have varied between 33-91%. The variableseroprevalence rates among these studies are likely due to markeddifferences in the size and demographics of the studies, and, perhapsmost importantly, differences in assay methods.

It is therefore desirable to implement a reliable and sensitive assayfor determining the presence of JCV antibodies that can be used, forexample, for assessing whether an individual has been exposed to JCV.

SUMMARY OF THE INVENTION

The invention relates to the development of an analytically validatedsensitive assay for detecting the presence of JCV antibodies in abiological fluid, serum or plasma.

Accordingly, the invention relates to a method that includes obtaining abiological sample from a subject (e.g., plasma, serum, blood, urine, orcerebrospinal fluid (CSF)); contacting the sample with highly purifiedviral-like particles (HPVLPs) under conditions suitable for binding of aJCV antibody in the sample to an HPVLP; detecting the level of JCVantibody binding in the sample to HPVLP; and correlating the detectedlevel with a reference, such that the reference is selected to indicatea false negative raw not greater than 3% and minimal cross reactivity toother components of the sample such as antibodies against other polyomaviruses, e.g. BK virus (BKV). In some embodiments, the reference,derived from a control sample or set of samples, is processed with thesample from the subject. In some embodiments, the reference is selectedsuch that the false negative rate of the assay is not greater than 1%.

In one embodiment, at least about 10% of the HPVLPs in a preparation ofpurified HPVLPs contain more than five VP1 polypeptides per HPVLP). Inother embodiments, at least about 15%, about 20%, about 25%, about 10%,about 40%, about 50%, about 60%, about 65%, about 70%, about 80% orabout 90% of the HPVLPs in a preparation of purified HPVLPs contain morethan five VP1 polypeptides per HPVLP.

The assay can be performed such that the HPVLP is immobilized on a solidsubstrate such as a microtiter plate or slide. In some embodiments, theHPVLP consists essentially of VP1 viral protein. The HPVLP can fartherinclude other viral proteins, for example at least one of a VP2 or aVP3. The viral protein(s) in the HPVLP can be recombinantly derived(e.g., a MAD1 strain VP1) or can be a naturally-occurring viral protein(e.g., derived from a naturally-occurring source). The method can beperformed using, for example, a biological sample obtained from asubject currently being heated with an immunomodulatory drug, a subjectconsidering initiating treatment with an immunomodulatory drug, or asubject suspected of having Progressive Multifocal Leukoencephalopathy(PML).

In some aspects, the assay method is a two-step assay that furtherincludes a secondary confirmation assay process that includes contactinga portion of the biological sample from the subject with HPVLP insolution (prior to incubating the sample with the HPVLP attached to asolid substrate), thereby providing a secondary sample; contacting thesecondary sample with HPVLP under the same conditions used for theprimary assay; detecting the level of JCV antibody binding to HPVLP inthe secondary sample; and comparing the detected level of JCV antibodyin the secondary sample to the level of JCV antibody in the sample thatwas not preincubated with soluble HPVLP, such that a decrease in thedetected level in the secondary assay sample compared to the sample thatwas not preincubated indicates the sample is positive for JCV antibody,and a change the detected level below a specified percentage indicatesthat there is no JCV-specific antibody present in the sample.

An assay described heroin can be used to assay for the presence of JCVantibodies in a subject who has never received treatment with animmunomodulator; or in a subject who has previously received animmunomodulator, but who is no longer receiving treatment with theimmunomodulator; or in subject who is presently undergoing treatmentwith an immunomodulator.

Detection of JCV antibodies binding to the HPVLPs in an assay featuredin the invention can indicate that a subject is at an increased risk forPML. Detection of JCV antibodies can also indicate that the subject isat an increased risk for adverse symptoms, such as the development ofPML, upon administration of certain therapeutic agents, such as certainimmunomodulators, and therefore be subject is not a candidate fortreatment with these agent. For example, detection of JCV antibodies insample from a subject can indicate that the subject is not a candidatefor treatment with an anti-VLA-4 therapeutic, such as natalizumab. Incertain embodiments, detection of JCV antibodies in a biological samplecan indicate that the subject is a candidate for treatment with animmunomodulator, such as natalizumab, except that the subject willundergo enhanced monitoring during treatment than a subject who does nothave detectable JVC antibodies. For example, the enhanced monitoring caninclude observation for adverse symptoms such as symptoms that mayindicate the development of PML.

Failure to detect JCV antibodies binding to HPVLPs in an assay featuredin the invention can indicate that the subject is a candidate to receivetreatment with an immunomodulator, such as natalizumab, and in oneembodiment, the subject is further administered the immunomodulator. Asubject determined not to have JCV antibodies can be re-tested at leastannually (e.g., at least every 3 months, every 6 months, every 9 months,or every 12 months) to determine whether the subject has developed JCVantibodies, which may indicate that the subject has been infected withJCV. A subject who previously did not have detectable JCV antibodies ina biological sample, and who subsequently develops XV antibodies in abiological sample, can stop receiving treatment with an immunomodulator.

In some embodiments, a subject who was previously identified as havingJCV antibodies, can be subsequently tested at a later date anddetermined not to have JCV antibodies. These subjects can be determinedto be candidates to receive treatment with an immunomodulator, such asnatalizumab. In one embodiment, a subject who previously tested positivefor the presence of JCV antibodies and who subsequently tested negativefor JCV antibodies can be administered the immunomodulator, and undergoenhanced monitoring as compared to a subject who never tested positivefor JCV antibodies, such as to monitor for symptoms that may indicatethe development of PML.

An assay featured in the invention is useful to treat a subject havingan immunological disease or disorder, such as multiple sclerosis (MS) orCrohn's Disease (CD). In one embodiment, an assay described herein hasbeen validated for use in MS and CD patients, such as by showing thatthe assay is effective to detect JCV antibodies in MS and CD patients ina controlled test environment, such as in a clinical trial.

In another aspect, the invention relates to a kit comprising an HPVLPand at least one reagent for performing an assay to identify a JCVantibody level in a sample, such as a biological sample.

In other aspects, the invention relates to a solution comprising HPVLPparticles consisting essentially of VP1-containing particles that aregreater in size than a VP1 pentamer, e.g., containing about 5, 10, 20,30, 40, 50, 60, 70 or 72 pentamers or containing about 25 VP1 molecules,about 50 VP1 molecules, about 100 VP1 molecules, about 150 VP1molecules, about 200 VP1 molecules, about 300 VP1 molecules, about 350VP1 molecules or about 360 VP1 molecules.

Another aspect featured in the invention is a method of preparing asolution of HPVLPs, the method comprising removing VP1-containingparticles from the solution that are the size of a VP1 pentamer or less.In one method VP1 polypeptides are expressed in cells, e.g., in insectcells or mammalian cells. The cells are lysed, and then the cells aretreated with a nuclease, such as BENZONASE®. Cell debris is removed byprecipitation, such as by salt (e.g., ammonium sulfate) precipitation,and then the VP1-containing supernatant is concentrated and furtherpurified using diafiltration, such as by one or two passages through amembrane, e.g., a tangential flow filtration (TFF) membrane. Thesolution containing the VP1-containing particles, e.g., HPVLPs, is thenfurther purified through an ion-exchange step, and elution of the HPVLPsis performed, e.g., with a buffer. VP1 purity can be assessed, e.g.,electrophoresis (e.g., SDS-PAGE) or mass spetometry. The presence ofHPVLPs can be confirmed by microscopy, e.g., electron microscopy. Thepercentage of total protein in the form of HPVLPs can be determined bysedimentation velocity analytical ultracentrifugation.

In one aspect, the invention features a method of identifying a subjectat risk of developing PML, such as by obtaining a biological sample fromthe subject; contacting the biological sample with HPVLPs underconditions suitable for binding of a JC Virus (JCV) antibody in thesample to an HPVLP; detecting the level of JCV antibody binding in thesample to HPVLPs; and correlating the detected level with a referenceset, wherein the subject is at increased risk of PML if JCV antibodybinding is detected. The reference set is selected to indicate a falsenegative rate of about 5%, about 3%, about 1% or less.

In another aspect, the invention features a method of identifying PMLrisk in a subject by determining the level of anti-JCV antibodies in asample from the subject, such as from a plasma, blood or serum sample;and assigning a risk level to the subject according to the level ofanti-JCV antibodies in the sample. The subject may be receiving animmunomodulatory therapy, such as an anti-VLA4 treatment, e.g.,natalizumab, or may be a candidate for receiving an immunomodulatorythereapy. In some embodiments, the subject has been diagnosed with animmunological disease or disorder, such as multiple sclerosis or Crohn'sdisease. In one embodiment, the level of anti-JCV antibodies isdetermined using a one-step assay, and in another embodiment, the levelof anti-JCV antibodies is determined using a two-step assay. Either theone-step assay or the two-step assay may include an ELISA assay.

In one embodiment, the method of identifying PML risk in a subjectfurther includes determining the level of anti-JCV antibodies in thesubject in a sample from a date subsequent to the initial sample;comparing the level of anti-JCV antibodies in the sample from thesubsequent date to the level in the sample from the initial sample; anddetermining whether the subject is at increased risk of PML at thesubsequent date compared to the time of the initial sample.

In one aspect, the invention features a method of monitoring PML risk ina subject, the method comprising determining the level of anti-JCVantibodies in a subject using a sample from a first date; assigning arisk of PML (e.g., high, or moderate or low risk) based on the level ofanti-JCV antibodies in the subject on the first date; determining thelevel of anti-JCV antibodies in the subject using a sample from a seconddate; and assigning a risk of PML (e.g., high, or moderate or low risk)based on the level of anti-JCV antibodies in the subject on the seconddate.

As used herein, an “HPVLP” is a highly purified VLP (“virus-likeparticle”) consisting predominantly of the VP1 protein. An “HPVLP”featured in the invention is composed mainly of the major capsid protein“VP1,” which can be a naturally-occurring VP1 or a recombinant VP1, fromthe polyomavirus, JC Virus (JCV). An HPVLP can be composed of, e.g.,more than one pentameric subunit, at least 10 pentameric subunits, atleast 20 pentameric subunits, at least 30 pentameric subunits, at least50 pentameric subunits, at least seventy-two pentameric subunits or moreof VP1. An HPVLP may contain VP1 polypeptides in an undeterminedconfiguration (e.g., the polypeptides may or may not be organized inpentamers), in which case an HPVLP can be composed of more than 5 VP1polypeptides, at least 50 VP1 polypeptides, at least 150 VP1polypeptides, at least 360 VP1 polypeptides or more. HPVLPs includecapsomeres, which contain about 10 to 24 pentamers. An HPVLP featured inthe invention can bind antibodies against naturally-occurring, intact JCvirus. In some embodiments, an HPVLP includes a second, and optionally athird, type of polypeptide that is a minor capsid protein of JC virus,e.g., at least one VP2 or VP3 polypeptide. The VP2 or VP3 can berecombinant or naturally-occurring or naturally-derived polypeptides.

Such “highly purified” particles contain more than one VP1 pentamer,e.g., at least 5, 10, 20, 30, 40, 50, 60, 70, 72 VP1 pentamers, or lessthan 100 VP1 pentamers. Such highly purified particles can be obtained,for example, by a method that involves double filtration. For example,in one embodiment, a highly purified preparation of VLPs is obtained bypurifying the particles at least twice by centrifugation, e.g., througha sucrose cushion. In general, an HPVLP preparation can be identified byits activity in an ELISA assay using defined control samples. In somecases, such control samples are negative controls and/or control samplescontaining low levels of JCV antibodies.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the results of an HPVLP ELISA on samplesfrom subjects positive for JCV DNA in their urine (Uropositive) andnegative for JCV DNA in their urine (Uronegative). The box representsthe interquartile (IQR) range with the median line in the center;brackets represent observations within 1.5 times the IQR. “+” signsrepresent observations beyond 1.5 times the IQR (outliers).*Mann-Whitney U test.

FIG. 2 is a graph depicting anti-JCV antibody levels as measured byELISA against urinary JCV DNA level as measured by qPCR (n=204). Opencircles represent urine and serum samples collected at matched STRATAtime points. Closed circles represent samples collected at differenttime points. For 17 samples with DNA test results below the level ofquantitation (<500 copies/mL) the level was set to the detection limit.

FIG. 3 is a graph depicting BKV-JCV cross-reactivity data from onerabbit immunized with BKV. Antisera from the BKV-immunized rabbit boundBKV VLPs with high affinity (EC50=1:100,000) and cross-reacted with JCVVLPs (EC50=1:5,000).

FIGS. 4A and 4B depict the anti-JCV assay reactivity of serum samplesfrom uronegative (n=311) (FIG. 4A) and uropositive (n=204) (FIG. 4B)patients in the screening and confirmation ELISAs. Distribution ofserological reactivity of the samples in the screening ELISA are shown,with lower (nOD₄₅₀=0.10) and upper (nOD₄₅₀=0.25) cut points highlighted(left panels). In the supplemental confirmation ELISA (right panels), a40% inhibition cut point is highlighted (vertical line) with shadedregions denoting samples that did not confirm to have anti-JCV specificantibodies (nOD₄₅₀≤0.25 and percent inhibition ≤40%).

FIGS. 5A and 5B are histograms depicting the frequency of observationswithin each 10% inhibition range for all patients (n=515) (FIG. 5A) anduropositive patients (n=204) (FIG. 5B). The distribution consisted oftwo clearly defined peaks, most optimally separated at 40% inhibition. A40% inhibition level corresponded to approximately the lower fifthpercentile of the response distribution of uropositive samples.

FIGS. 6A and 6B are plots of nOD₄₅₀ values from the screening ELISA(FIG. 6A) versus percent inhibition values from the confirmation ELISA(FIG. 5B) for the 11 pre-PML samples. Horizontal lines represent nOD₄₅₀values of 0.10 and 0.25, the vertical line represents percent inhibitionof 40%.

DETAILED DESCRIPTION OF THE INVENTION

A sensitive assay for JCV antibodies that minimizes false negatives andminimizes detection of cross-reacting antibodies is useful foridentification of individuals that have been exposed to JCV. Deploymentof such a test may be useful in the identification of individuals whohave a current JCV infection or have had sufficient past exposure to JCVto develop antibodies against the virus. Such an assay may also providea tool to assist clinicians with PML clinical vigilance and riskstratification. For example, such a test may be useful for practitionersand patients as part of an evaluation of a patient's risk of developingPML by accurately assessing whether a subject has been exposed to JCV.In some cases, the analysis may include determining JCV antibody levelsin a biological sample from the patient.

Certain difficulties lie in development of a useful assay for JCVantibodies, for example, the establishment of validated cut points.Applicants have solved this problem using data derived from assays ofurine and plasma samples from patients that are uropositive oruronegative for JCV DNA. Another problem is developing an assay withspecificity and reproducibility. Applicants have solved this problem byusing a highly purified viral protein-containing particle in an antibodyassay. In addition, applicants have discovered that the use of asecondary assay to resolve samples with ambiguous results in the primaryassay improves the utility of the assay for providing a useful resultfor such samples.

Accordingly, an analytically validated assay that uses a highly purifiedVP1-containing virus-like particle (VLP) has been developed to detectthe presence of JCV antibody in a body fluid, such as serum, plasma,urine, CSF, or other body fluid that contains antibodies. In experimentsto validate the new assay, an approximately 54% prevalence of JCVantibodies in a population of MS patients enrolled in a clinical studywas identified. A key feature of the assay described herein is the useof a highly purified viral-like particle (HPVLP).

One advantage of the assay described herein is that it has a relativelylow false negative rate, e.g., a false negative rate of about 10%, about8%, about 6%, about 4%, about 3%, about 1% or less for the detection ofantibodies to JCV. In general, the assay has a false negative rate ofonly about 3% or less for the detection of antibodies to JCV. Asdescribed herein the new assay can be used to monitor the seroconversionrate for JCV. For example, the assay has been used to discover an annualseroconversion rate of no more than about 2% in a tested cohort ofsubjects who were initially negative for JCV antibody. This demonstratesthat the assay can be useful for monitoring the JCV exposure status ofan individual over time.

The assay can be used for the detection of JCV antibodies in any humansubject, including a subject considering treatment with animmunomodulator, for example an anti-VLA-4 therapy (e.g., natalizumab),an anti-CD20 therapy (e.g., rituximab), an anti-CD11 a therapy (e.g.,efalizumab), or mycophenolate mofetil; in a subject currently beingtreated with an immunomodulator; or a subject that has ceased treatmentwith an immunomodulator. The assay may be useful to others who may besusceptible to PML, such as individuals having lymphoproliferativedisorders, such as multiple myeloma or a lymphoma; individuals infectedwith human immunodeficiency virus (HIV), or having acquired immunedeficiency syndrome (AIDS), hematologic malignancies, or an autoimmunedisease such as systemic lupus erythematosus (SLE), an inflammatorybowel disease, such as Crohn's Disease (CD) or ulcerative colitis,multiple sclerosis (MS) or arthritis, e.g., rheumatoid arthritis (RA).The assay may also be useful to subjects receiving immunosuppressive orimmunomodulatory therapies, such as transplant patients. Exemplaryimmunosuppressive or immunomodulatory therapies include natalizumab,rituximab, efalizumab, and mycophenolate mofetil. The assay can beuseful for detection of JCV antibodies in a subject having a disorder,or being treated with a drug, disclosed in Piccinni et al. “Strongerassociation of drug-induced progressive multifocal leukoencephalopathy(PML) with biological immunomodulating agents” Eur. J. Clin. Pharmacol.66:199-206, 2010, the contents of which are incorporated herein byreference.

VP1

It was discovered that the use of HPVLPs in an assay for JCV antibodiescan improve the accuracy of the assay and is useful in an assay suitablefor analytic and diagnostic purposes. VP1 for use in producing HPVLPscan be generated using methods known in the art and can be eithernaturally-occurring VP1 or recombinantly produced VP1, e.g., a VP1 froma JCV virus. In general, the VP1 used is VP1 from MAD1 strain of JCV. Insome embodiments, the VP1 used in the assay comprises VP1 from more thanone JCV strain, for example, from one or more of strains 1A, 1B, 2A, 2B,3, 4, and 7. After preparation of VP1, e.g., recombinantly synthesizedVP1, the VP1 for use in the assays described herein is then furtherpurified through standard biochemical methods includingdensity-gradient/ultracentrifugation methods, or a series of chemicalprecipitation steps, concentration/diafiltration and ion-exchangechromatography. The purification methods typically include a step toremove smaller proteins including monomer VP1 polypeptides, or pentamerVP1. The removal of these smaller particles can be done in, for example,in one step or in two steps (e.g., a first filtration step to remove VP1monomers, and then a second filtration step to remove pentamer VP1particles). Such biochemical purification methods are known to those inthe art. Examples 1 and 7 provide two different methods of JCV VP1-VLPpurification.

An HPVLP preparation (HPVLPs) according to one aspect of the presentinvention does not contain significant amounts of VP1 monomer (e.g., hasbeen purified to remove monomers). An HPVLP preparation according toanother aspect of the present invention does not contain significantamounts of VP1 molecules in configurations the size of a VP1 pentamer,or smaller (including monomer). The HPVLP can be prepared fromrecombinant VP1 or naturally-occurring VP1 (e.g., isolated from virus orvirus capsid). In some embodiments, additional JCV components, such asone or both of the minor coat proteins from JC virus, e.g., VP2 or VP3,are included in the HPVLP particle or are associated with the substrate.

In some cases, recombinantly expressed VP1 may not assemble intopentamers or HPVLPs that resemble naturally-occurring viral capsids, forexample, recombinantly expressed VP1 may assemble into tubes or othernon-spherical geometries. Accordingly, the invention relates to methodsof producing HPVLPs that are substantially spherical in geometry. Theinvention includes HPVLP preparations where at least about 10%, about15%, about 20%, about 25%, about 50%, about 60%, about 65%, about 70%,about 80%, about 90%, about 95%, or about 99% of the HPVLPs in thepreparation resemble the naturally-occurring JCV capsid (e.g., are in anicosahedral or substantially spherical configuration). In someembodiments, an HPVLP preparation contains at least 10%, 15%, 20%, 50%,60%, 70%, 80%, 90%, 95%, or 99% of the HPVLPs in the preparationresemble the naturally-occurring JCV capsid. Such methods can includeexpressing viral proteins under conditions that result in such apreparation and/or isolating and purifying expressed viral proteins asdescribed herein to produce such a preparation.

Methods of making HPVLP

HPVLPs can be made, for example, by transforming a baculovirus with avector expressing a VP1 gene, such as a VP1 gene from a JC virus. Thebaculovirus is used to infect a cell culture, such as an insect cellculture (e.g., SF9 cells) or a mammalian cell culture, and the cellsexpress the VP1 protein. HPVLPs are isolated by lysing the cells, andpurifying the particles through a series of centrifugation andultrafiltration steps. In general, the purification is performed usingmethods such as sucrose cushion sedimentation, isopycnicultracentrifugation and extensive ultrafiltration or other methods knownto those in the art. In certain embodiments, the purification willinclude twice centrifuging the particles through a sucrose cushion. Inan alternative purification method, cells are lysed, and particles areisolated by a series of precipitation and concentration/diafiltrationsteps with a final ion-exchange step.

Purity can be assessed using any suitable techniques known in the art,for example, analytical ultracentrifugation, electron microscopy, PAGEanalysis, mass spectrometry, protein concentration, or activity in anELISA with control sera. Insufficiently purified VLPs result in a highbackground yielding falsely high JCV antibody levels or calculatedexposure rates.

In some embodiments, the HPVLPs contain VP1 as the sole JC virusprotein.

In some embodiments, the HPVLPs are heterogeneous particles, andtherefore include VP1 protein, and at least one of the minor coatproteins of JC virus, e.g., VP2 or VP3. In another embodiment, the HPVLPincludes VP1, VP2 and VP3 proteins. An HPVLP that includes VP1 and VP2can be produced using methods known in the art, for example, bytransforming a baculovirus with a nucleic acid including a VP1 and a VP2gene, such as under the control of the same or different promoters. Acell culture is infected with the baculovirus, and the cells express VP1and VP2, and HPVLPs form which include both types of proteins. In oneembodiment, the VP1 and VP2 genes are on different DNA molecules, theDNA molecules are transformed into different baculoviruses and thebaculoviruses are used to transfect cells in the same culture. The cellsexpress the VP1 and VP2 proteins, and HPVLPs form which include bothtypes of protein. In some cases, a heterogeneous HPVLP will include,e.g, one or two VP2 polypeptides for every five VP1 polypeptides. Ingeneral, an HPVLP will contain more VP1 polypeptides than VP2polypeptides, as is the case in naturally-occurring JC virus.

An HPVLP that includes both VP1 and VP3 or both VP1 and VP2 moleculescan be produced, for example, by transforming a baculovirus with anucleic acid including a VP1 and a VP3 gene or a VP1 and VP2 gene,respectively, under the control of the same or different promoters. Acell culture is infected with the baculovirus, and the cells express VP1and VP3 or VP1 and VP2, and HPVLPs form which include both types ofproteins. In some embodiments, the VP1 and VP3 or VP1 and VP2 genes areon different DNA molecules, the DNA molecules are transformed intodifferent baculoviruses, and the baculoviruses are used to transfectcells in the same culture. The cells express the VP1 and VP3 proteins orVP1 and VP2 genes, respectively, and HPVLPs form which include bothtypes of protein. HPVLP particles can be isolated from such preparationsusing methods known in the art such as those used to isolate JCVcapsids.

Typically, a VP1 pentamer that is in a heterogeneous HPVLP will include,e.g, five VP1 polypeptides and one VP3 polypeptide and/or one VP2polypeptide, depending on whether a VP3 gene or VP2 gene was used tomake the constructs. There will typically be more VP1 polypeptides thanVP3 or VP2 polypeptides in an HPVLP. In some embodiments, the VP2 or VP3is from a polyoma virus that is not a JC virus, e.g., a BK viruspolypeptide.

An HPVLP that includes all three of VP1 and VP2 and VP3 molecules can beproduced by transforming a baculovirus with a nucleic acid (e.g., acircular DNA, e.g., <5.5 kb) including a VP1, VP2 and VP3 gene, such asunder the control of the same or different promoters. A cell culture,such as a mammalian cell culture, is infected with the baculovirus, andthe cells express VP1, VP2 and VP3 proteins. HPVLPs consequently formwhich include all three types of proteins. In one embodiment, the VP1,and either or both of the VP2 and VP3 genes are on different DNAmolecules, the DNA molecules are transformed into the same or differentbaculovirus, and the baculovirus are used to infect cells in the same orseparate cultures. The cells express the VP1, VP2 and VP3 proteins, andHPVLPs form which include both types of protein. A heterogeneous HPVLPcan include, e.g, five VP1 polypeptides and one each of VP2 and VP3polypeptides, although the ratios may vary within a preparation. Therewill typically be more VP1 polypeptides than VP2 and VP3 polypeptides inan HPVLP.

In some embodiments, the HPVLP will be greater in size than a VP1pentamer. By greater in size, it is meant that the mass of proteincontained in an HPVLP particle is greater than a pentamer containingsolely VP1.

In other embodiments, the method of preparing a solution of HPVLP caninclude removing from the solution particles (e.g., VP1 monomers orsmall VP1 containing particles) that are the size of a VP1 pentamer orsmaller. Methods such as centrifugation and size-exclusionchromatography can be used to perform this purification step. In someembodiments, other methods known in the art, e.g., ion exchangechromatography, can be used in the preparation of HPVLPs that are largerthan a VP1 pentamer. In general, an HPVLP preparation suitable for usein an assay will contain at least 20% HPVLPs, at least 25% HPVLPs, atleast 40% HPVLPs, at least 60% HPVLPs, at least 65% HPVLPs, at least 70%HPVLPs, at least 80% HPVLPs, at least 85% HPVLPs, at least 90% HPVLPs,at least 95% HPVLPs, or at least 99% HPVPLs compared to non-HLVLPparticles (e.g., by percent of pentamers compared to VP1 monomers andaggregates containing fewer than five VP1 molecules).

Cut point

The invention provides methods of analysis that employ “cut points” toreduce false negative and false positive rates. The cut points areestablished based on data from the HPVLP assays (e.g., to detect JCVantibodies in a biological sample), averaged, for example, betweenduplicate test samples and multiple replicates (for example, at leasttwo, at least four, or at least eight replicates of control samples).

In one version of an assay according to the present invention, resultsfrom initial HPVLP screening assays, e.g., ELISA assays, will cause atest sample to be classified as having or not having JCV-specificantibodies, or, if the sample does not fall under one of these twoclassifications, then the sample will be subjected to a supplementalconfirmation assay. For example, samples that produce a result in anHPVLP ELISA assay featured in the invention less than an establishedlevel (e.g., an nOD₄₅₀<0.1) will be classified as lacking JCV-specificantibodies, and samples that provide a result in the ELISA greater thanan established level (e.g., an nOD₄₅₀>0.25) will be classified aspositive for JCV-specific antibodies. Samples that do not clearly fallinto one of these classifications (e.g., 0.1<OD450<0.25) can be testedin a confirmatory assay.

In one embodiment, the confirmatory assay requires a pre-incubationstep, where the test sample is pre-incubated with buffer (or othersuitable solution) control or with HPVLPs (in buffer or other suitablesolution) to pre-adsorb JCV-specific antibodies prior to analysis in anHPVLP ELISA, as described in further detail below. After pre-incubationwith HPVLP if the reaction in the primary assay decreases by less than40% compared to buffer control, then the sample is interpreted to benegative for the presence of JCV-specific antibodies. If the resultsshow a ≥40% reduction in reaction compared to buffer control in theprimary assay after pre-incubation with HPVLP then the sample isinterpreted to contain JCV specific antibodies. In some embodiments,only the confirmatory assay is performed.

An example of a method for selecting and verifying suitable cut pointsis provided in Example 4.

Substrate

Any suitable solid substrate can be used for the HPVLP assay format. Insome embodiments, the substrate is a microtiter plate (e.g., a 96-wellplate) a slide, a bead, or a column. The substrate can be suitable forchromogenic or chemiluminescent detection methods.

Assay

Assays are conducted by adding a biological sample to a substrate thathas been coated with an HPVLP and detected using methods known in theart. In general, a solid base platform is used such as a microtiterplate (for example, a 96 well plate); although other formats known inthe art can be used. In some embodiments, the biological sample isdiluted prior to use in an assay.

In certain embodiments, the assay format is an enzyme-linked immunoassay(ELISA). Broadly, the method typically includes coating the substratewith capture antigen such as HPVLP, incubating sample containing bindingantibodies directed to capture reagent, washing to removenon-specifically bound species, and detecting the bound immunecomplexes, e.g., by a chromogenic or chemiluminescent assay. Chromogenicsubstrates produce a colored end product, which can be detected andmeasured visually or with the use of a spectrophotometer.Chemiluminescent substrates produce light, which can be measured using aluminometer.

Coating a plate with HPVLP generally includes incubating the solidsubstrate (such as wells of a microtiter plate) with a solution of HPVLPat a suitable concentration (e.g., 1 μg/ml), either overnight or for aspecified number of hours. The HPVLP can include VP1 as the only JCVviral component, or the HPVLP can be a heterologous particle, thatcontains at least one of VP2 or VP3 per particle or at least one each ofVP2 and VP3 per particle. After coating with the HPVLP, the wells of theplate are washed. The substrate is then “coated” with a nonspecificprotein that is antigenically neutral with regard to the samples to betested. Suitable coating materials are known in the art and includebovine serum albumin (BSA), casein or solutions of milk powder.

The sample or reference is incubated on the prepared substrate underconditions effective to permit complex formation (HPVLP/JCV antibody),thus forming a bound complex. Detection of the bound complex isperformed using a labeled antibody that can bind to human antibody. Ingeneral, the labeled antibody can detect human IgG or human IgG and IgM.In some cases, the assay can be performed using secondary or tertiarydetection methods.

A reference sample can be of the same biological material (e.g., plasma,serum, urine, or CSF) isolated from an individual known to be infectedwith JC virus based on the presence of JCV DNA in urine of theindividual (uropositive). A reference sample is used to establish theassay cut point such that the false negative rate of the assay is notgreater than 1%-3%.

“Under conditions effective to permit complex formation” generally meansconditions in which the reagents have been diluted to reduce backgroundand provide readouts of results that lie within a specified range.Diluents can include, in non-limiting examples, solutions that includeBSA, phosphate buffered saline (PBS), or PBS containing TWEEN®.

“Suitable” conditions also include conditions that are at a temperatureand/or for a period of time sufficient to allow effective binding.Incubations are typically from about one to two hours or one to fourhours, at temperatures of approximately 25° C. to 27° C., or may beovernight at about 4° C. However, those in the art will understand thatother conditions may be suitable.

In general, one or more washes are conducted between the incubations ofthe assay. Appropriate wash solutions include diluent buffer (e.g., PBSor PBS/TWEEN®) or borate buffer.

In general, the detection of antibody bound to HPVLP is performed usingmethods well known in the art. In general, such methods are based on thedetection of a label or marker, such as a radioactive, fluorescent,biological or enzymatic tag. U.S. patents concerning the use of suchlabels include, for example, U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. In general,the detection of JCV antibody binding is detected using a secondaryantibody that is labeled. In general, the secondary antibody is specificfor detecting human IgG. Quantification is achieved by measuring thedegree of color generated, e.g., using a visible spectraspectrophotometer.

Example 2 illustrates a method of performing the assay and those in theart will understand that suitable modifications can be made.

In one embodiment, the assay is performed in a medical office, such asby a healthcare provider, e.g., a doctor, a nurse or a technician,working in a facility where the biological sample is obtained from apatient. In another embodiment, the biological sample obtained from apatient is transported to another facility, e.g., to a third partyfacility, where the assay is performed. In this latter case, the resultsof the assay can be reported back to the healthcare provider, such asthrough a form, which can be submitted by mail or electronically (e.g.,through facsimile or e-mail) or through an on-line database. In oneembodiment, the results of the assay (including the screening assay and,optionally, a confirmatory assay) can be stored in a database and can beaccessed by a healthcare provider, such as through the worldwide web.

Secondary Test

In some cases, for example, when the level of JCV antibody in a samplefalls into a designated “equivocal zone” or “indeterminate zone,” e.g.,where it is determined that there is limited certainty regarding thepresence or absence of JCV antibody, a secondary test (also referred toherein as a “confirmatory assay”) of the sample is employed. For thesecondary test, two aliquots of a biological sample are used. The firstis prepared prior to use in the assay by preincubating the sample in thepresence of assay buffer in solution for a period of time (e.g., for 30minutes, one hour, or longer such as overnight at 4° C.). The secondaliquot is prepared prior to use in the assay by preincubating thesample in the presence of HPVLP in solution for a period of time (e.g.,for 30 minutes, or one hour or longer). The two aliquots are then usedin the HPVLP assay as described herein, and the assignment of the sampleto JCV antibody positive or antibody negative is made. If the assayresults for the aliquot incubated with HPVLP in solution is the same asfor the first aliquot incubated with buffer in the primary assay (i.e.,approximately the same OD), then the sample is interpreted to benegative for the presence of JCV-specific antibodies. If the assayresults are lower after pre-incubation (i.e., in the secondary assay),then the sample is interpreted to contain JCV specific antibodies.

An assay featured in the invention that utilizes a secondary test isalso referred to herein as a “two-step test” or a “two-step assay.”

Reporting of Assay Results

In some embodiments, the assay includes a read out that can be a level(e.g., OD) relative to a reference or a read out that is an evaluationof whether the sample is positive, negative, or indeterminate for thepresence of JCV antibodies. In some embodiments, a kit is provided thatincludes at least HPVLP and optionally, other components for an assay.For example, the kit can include assay positive and negative controls,buffers and substrates (e.g., microtiter plates) for preparing the toolsto perform the primary ELISA assay, and the secondary confirmationassay. The kit can include, e.g., solvents or buffers, controls, astabilizer, a preservative, a secondary antibody, e.g., an anti-HRPantibody (IgG) and a detection reagent.

The HPVLP can be provided in any form, e.g., liquid, dried, semi-dried,or lyophilized form, or in a form for storage in a frozen condition. Insome embodiments, prepared HPVLPs are pelleted and stored in asemi-solid form.

Typically, HPVLPs are provided in a form that is sterile. When HPVLP isprovided in a liquid solution, the liquid solution generally is anaqueous solution, e.g., a sterile aqueous solution. When the HPVLP isprovided as a dried form, reconstitution generally is accomplished bythe addition of a suitable solvent. The solvent, e.g., sterile buffer,can optionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining HPVLPs in a concentration suitable for use in the assay orwith instructions for dilution for use in the assay. In someembodiments, the kit contains separate containers, dividers orcompartments for the HPVLP and assay components, and the informationalmaterial. For example, the HPVLPs can be contained in a bottle or vial,and the informational material can be contained in a plastic sleeve orpacket. In other embodiments, the separate elements of the kit arecontained within a single, undivided container. For example, an HPVLPcomposition is contained in a bottle or vial that has attached theretothe informational material in the form of a label. In some embodiments,the kit includes a plurality (e.g., a pack) of individual containers,each containing one or more unit forms (e.g., for use with one assay) ofHPVLP. For example, the kit includes a plurality of ampoules, foilpackets, or blister packs, each containing a single unit of HPVLP foruse in a screening or confirmatory assay. The containers of the kits canbe air tight and/or waterproof. The container can be labeled for use.

In one embodiment, the kit can include informational material forperforming and interpreting the assay. In another embodiment, the kitcan provide guidance as to where to report the results of the assay,e.g., to a treatment center or healthcare provider. The kit can includeforms for reporting the results of an HPVLP assay described herein, andaddress and contact information regarding where to send such forms orother related information; or a URL (Uniform Resource Locator) addressfor reporting the results in an online database or an online application(e.g., an app). In another embodiment, the informational material caninclude guidance regarding whether a patient should receive treatmentwith an immunomodulatory drug, depending on the results of the assay.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about HPVP assayand/or its use in the methods described herein. Of course, theinformational material can also be provided in any combination offormats.

In some embodiments, a biological sample is provided to an assayprovider, e.g., a service provider (such as a third party facility) or ahealthcare provider, who evaluates the sample in an assay and provides aread out. For example, in one embodiment, an assay provider receives abiological sample from a subject, such as a plasma, blood or serumsample, and evaluates the sample using an assay described herein, anddetermines that the sample contains JCV antibodies. The assay provider,e.g., a service provider or healthcare provider, can then conclude thatthe subject is at increased risk for PML. The assay provider can furtherdetermine that the subject is not a candidate to receive treatment withan immunomodulator, such as an anti-VLA therapy, such as natalizumab, orthat the subject is a candidate to receive treatment with animmunomodulator, but the candidate will have enhanced monitoring ascompared to a subject who is determined not to have JCV antibodies. Forexample, the candidate will be examined more frequently for thedevelopment of adverse symptoms, such as symptoms that may indicate thedevelopment of PML.

In one embodiment, the assay provider performs an assay described hereinand determines that a subject does not have detectable JCV antibodies.The assay provider further determines that the subject is a candidate toreceive treatment with an immunomodulator, such as natalizumab. In oneembodiment, the assay provider informs a healthcare provider that thesubject is a candidate for treatment with the immunomodulator, and thecandidate is administered the immunomodulator.

The assay provider can provide the results of the evaluation, andoptionally, conclusions regarding one or more of diagnosis, prognosis,or appropriate therapy options to, for example, a healthcare provider,or patient, or an insurance company, in any suitable format, such as bymail or electronically, or through an online database. The informationcollected and provided by the assay provider can be stored in adatabase.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

Examples Example 1: Synthesis and Purification of Highly Purified VP1Particles

HPVLPs consisting of JCV or BKV capsid protein VP1 were produced in SF9insect cells transfected with a recombinant baculovirus. In the case ofJCV VP1 containing particles, recombinant baculovirus was transformedwith a nucleic acid expressing VP1 from the Mad-1 strain of JCV. Therecombinant VLP was harvested prior to cell lysis and was purified bydifferential ultracentrifugation, detergent washing and ultrafiltration.

Briefly, baculovirus infected cells were harvested about three days postinfection by centrifugation at 3000×G and stored frozen untilpurification of HPVLPs. Purification was performed using about 100 gramsof frozen cell pellets. Thawed cells were lysed in 500 ml of PBSsupplemented with 0.1 mM CaCl₂ (PBS-C). The cells were disrupted bypassing the cell suspension twice through a MicrofluidicsMicrofluidizer®. Cell debris was removed by pelleting at 8000 ×G for 15minutes. The supernatant volume was adjusted to 720 ml with PBS-C andloaded onto 5 ml 40% sucrose cushions. HPVLPs were twice pelletedthrough the sucrose cushions in a SW28 rotor at 100,000×G for 5 hours.The HPVLP pellets were resuspended in PBS-CaCl₂) and then treated with0.25% deoxycholate for 1 hour at 37° C. followed by the addition of 4 MNaCl supplemented with 0.1 mM CaCl₂) for 1 hour at 4° C. Precipitatedmaterial was removed by centrifugation at 8000×G for 15 minutes. Theresulting supernatant was concentrated and buffer exchanged byultrafiltration through a PELICON®-2 500,000 MWCO membrane (Millipore).The concentrated VLPs were applied to the center of a 25-40% stepgradient of Optiprep™ (Sigma, St. Louis, Mo.) and banded at 190,000 gfor 17 hours in a Type 50.2 rotor. VLP bands were collected and thenconcentrated and buffer exchanged in an AMICON® stirred cell (Millipore)with a 300,000 MWCO (molecular weight cut-off) membrane. Theconcentrated material was filtered through a 0.22 μPES(polyethersulfone) filter and stored at 4° C. VLPs prepared in this wayare termed HPVLPs herein. VLP quality is generally determined by gelelectrophoresis and electron microscopy.

To denature the VLPs for protein determination, EDTA, DTT and SDS wereadded to final concentrations of 2 mM, 2 mM and 2% respectively. Theconcentration of the fully denatured protein was determined by using thePIERCE′ BCA (bicinchoninic acid) assay.

For analysis by gel electrophoresis, a sufficient volume to give 2 μg to5 μg of total protein was loaded on precast 4% to 20% polyacrylamidegels (NOVEX, San Diego, Calif.) by using a NuPAGE®morpholineethanesulfonic acid-SDS buffer system (Invitrogen, Carlsbad,Calif.). The gels were electrophoresed at a constant current of 70mA/gel to 80 mA/gel for 30 minutes. Protein bands were fixed with 50%methanol and 10% acetic acid in distilled water and visualized with acommercial colloidal Coomassie blue reagent (Invitrogen) according tothe recommendations of the manufacturer.

VLPs were evaluated using electron microscopy. VLP samples were placedon carbon grids, briefly washed in water and negatively stained withuranyl acetate and allowed to dry. The grids were viewed and imaged on aTecnai™ G2 Spirit BioTWIN TEM.

An alternative JCV VP1-VLP purification method is presented below, atExample 7.

Example 2: HPVLP Antibody Assay

A sensitive assay for anti-JCV antibodies was developed using the HPVLPsdescribed herein and is referred to herein in its various embodiments asan HPVLP assay. In an example of the assay, 96 well microtiter plateswere prepared by adding a solution containing HPVLP at a concentrationof 1 μg/ml and incubating the plate overnight at 4° C. The wells wererinsed with diluent buffer and then blocked for one hour at roomtemperature with Casein Blocking Buffer and rinsed with diluent buffer.The assay controls and serum or plasma samples were diluted 1:200 inassay diluent. The diluted samples and controls were added to wells andincubated for one hour at room temperature and washed with diluentbuffer. Detection was performed using donkey anti-human-HRP antibody(IgG), which was added to the wells and incubated at room temperaturefor one hour. Plates were then washed and TMB(3,3′,5,5′-tetramethylbenzidine) buffer (Chromagen, Inc., San Diego,Calif.) was added. After a development for a time suitable to permitcolor to develop (about 20 minutes), the reaction was stopped with 1 NH₂SO₄, and the absorbance at 450 nm was read. Levels of anti-JCVantibody in the samples were expressed as OD units.

The assay was interpreted as described below using the OD units todetermine levels.

In secondary testing, if unknown samples produced greater than 40%competitive inhibition of binding with HPVLP in solution, the sample wasconsidered JCV+(JCV positive), with <40% inhibition being scored asJCV−(JCV negative).

Initially, samples with OD values greater than the cut point OD (meanNegative Control OD×1.23) were defined as positive for the presence ofJCV antibodies, whereas samples with OD values equal to or less than thecut point OD were defined as negative.

Controls used in the assay were selected based on target OD andspecificity (as determined in the secondary confirmation assay forspecificity (described infra) and included Positive Control 1, which waspooled donor sera with high reactivity in the assay defined as havingtarget OD value of about 1.0 and for specificity, competed withJCV >80%; Positive Control 2, which contained pooled donor sera withlower reactivity in assay defined as having a target OD value of about0.25 in the assay; and for specificity competed with JCV >80%; andNegative Control, which was pooled donor sera with reactivity similar tobuffer control in assay having a target OD value of approximately 0.07(note that the assay buffer has an O.D. value of approximately 0.045).

In some cases, a titration assay was conducted in which positive sampleswere tested at multiple dilutions, and the highest dilution giving an ODvalue greater than the cut point OD was defined as the JCV IgG titer.

The assays have been validated from the perspective of specificity,precision, matrix interference, robustness, and reagent stability.

Example 3: Secondary Confirmation Assay

In some cases, a secondary confirmation assay (secondary assay) wascarried out in addition to the test described supra. In the confirmationassay, samples (plasma or serum) were incubated with HPVLP (final VLPconcentration=1 μg/mL; final sample dilution=1:200) for one hour at roomtemperature prior to use in the assay. Control samples were incubated inassay buffer, and not in the presence of HPVLP. The assay was thenconducted as described above. A percent nOD₄₅₀ inhibition was calculatedas: % inhibition=100 ×[1- (average nOD₄₅₀) (JCV MAD-1 VLP pre-incubatedsamples) ÷(average nOD₄₅₀) (buffer incubated samples)].

If the assay results were the same after pre-incubation with buffer asin the primary assay (i.e., approximately the same O.D.), then thesample was interpreted to be negative for the presence of JCV-specificantibodies. If the assay results were lower after pre-incubation withHPVLPs (i.e., in the secondary assay), then the sample was interpretedto contain JCV-specific antibodies.

Example 4: Screening/Confirmation Assay Cut Point Algorithm

The serological test (JCV antibody test) was configured as a two-stepassay: a screening ELISA and a supplemental confirmation ELISA(secondary assay).

For comparison of results between assay plates, assay runs, andanalysts, sample results were normalized to the optical density (OD₄₅₀)value of the positive control on the plate and reported as normalizedOD₄₅₀ as described below.

To implement the utility of the HPVP assay, cut points were derivedusing a Weibull three component mixture-distribution model. In thesedeterminations, the following definitions were used:

${{{Screening}\mspace{14mu}{assay}\mspace{20mu}{normalized}\mspace{14mu}{OD}\mspace{14mu}({nOD})} = \frac{{avg}\;\left( {{sample\_ OD}{\_ duplicates}} \right)}{{avg}\;\left( {{PC}\; 1{\_ OD}{\_ replicates}} \right)}};$

For example:

Average (sample_OD_duplicates)=0.60

Average (Positive Control 1 OD_replicates)=1.20

Normalized OD=0.60/1.20=0.50.

For the Confirmation Assay

${{Confirmation}\mspace{14mu}{assay}\mspace{14mu}\%\mspace{14mu}{inhibition}} = {100\% \times \left( {1 - \frac{{competition\_ sample}{\_ OD}}{{noncompetition\_ sample}{\_ OD}}} \right.}$

In the supplemental confirmation ELISA, soluble HPVLP was used topre-adsorb high affinity antibodies against JCV in samples prior toevaluation of the samples in the screening ELISA. Results werecalculated as percent inhibition to determine decreases in reactivity inthe screening ELISA after the samples were pre-adsorbed with HPVLP [%inhibition=100 ×[1- (average nOD₄₅₀ HPVLP pre-incubated samples)÷(average nOD₄₅₀ buffer incubated samples)].

False positive and false negative rates were defined as follows. Thefalse negative rate is the proportion of true JC virus positive samplesthat are determined to be antibody negative by the assay. Thesero-positive rate is the proportion of samples determined to besero-positive (i.e., have JCV antibodies as determined using theanti-JCV screening/confirmation cut point algorithm).

Data were analyzed using SAS v9. Data not demonstrating a normaldistribution were analyzed by the Mann-Whitney U test. Categorical datawere analyzed using Pearson's χ2 test or Fisher's exact test dependingon the sample size. Pearson's correlation coefficient was used to assesthe relationship between nOD₄₅₀ and urinary JCV DNA levels. All testswere two-sided at an alpha level of 0.05. Confidence limits for theseroprevalence and false-negative rates were obtained by the bootstrappercentile method (6) using 10,000 bootstraps.

Example 4(a): Serological Reactivity to JCV

A study was conducted to establish an assay to detect anti-JCVantibodies in MS patients and to conduct a preliminary evaluation of thepotential clinical utility of the assay for PML risk stratification. Tocharacterize antibody responses against infectious agents in humans, itwas critical to have reference sera from both infected and non-infectedindividuals. While the asymptomatic nature of JCV infection makes itimpossible to identify “true” negative individuals, Applicants were ableto identify a population of “true” positive individuals by measuring JCVDNA in the urine of “uropositive” individuals.

Urinary JCV DNA levels (collected in the STRATA (natalizumabreinitiation of dosing) clinical trial protocol) were determined by aquantitative real-time polymerase chain reaction (q-PCR) assay (ViraCorLaboratories, Lee's Summit, MO) with a limit of quantitation of 500copies/mL and a limit of detection of 50 copies/mL.

The anti-JCV antibody status of 831 MS patient serum samples, whichincluded samples from 204 JCV uropositive patients, was initiallyevaluated for anti-JCV antibodies in a screening ELISA to determine thedistribution of serological responses. The assay results by urinary DNAstatus showed the presence of two overlapping yet distinct populationsof JCV IgG reactivity (FIG. 1). The median level of reactivity for JCVDNA uropositive MS patients (nOD₄₅₀=0.895) was significantly higher thanfor JCV DNA uronegative MS patients (nOD₄₅₀=0.131; p<0.001), and nouropositive patient showed assay reactivity below a nOD₄₅₀ of 0.10.Therefore, a lower assay cut point was established at nOD₄₅₀ 0.10,wherein the empirical false-negative rate in the negative zone was 0%.

Many patients with no detectable JCV DNA in the urine (uronegatives) hadserological reactivity similar to that of uropositive patients. Theseresults are consistent with the assumption that a urine JCV DNA test islikely to fail to detect all JCV infected individuals.

Example 4(b): Urinary JCV DNA Load and Serological Activity

To address the potential concern that JCV infected patients with lowlevels of viral replication may have low serum antibody levels that arenot detected in the serological assay (potential false negatives) thecorrelation between viral levels and antibody reactivity were examined.FIG. 2 shows data from the 204 JCV DNA uropositive STRATA patients, andillustrates that there is no detectable relationship between urinary JCVDNA levels and anti-JCV antibody levels in samples with nOD₄₅₀ below0.60 (Pearson's correlation coefficient=0.048, p=0.751). This resultholds true even if the urine and serum were collected at the same STRATAstudy time point (Pearson's correlation coefficient=0.002, p=0.993). AtnOD₄₅₀>0.60, a stronger correlation was observed with a higherproportion of serum samples from individuals with high JCV DNA copies/mLexhibiting higher nOD₄₅₀ values, consistent with literature reports(e.g., Egli et al., J. Infect. Dis. 199:837-846, 2009). These datasuggest that seronegative results are likely due to an absence of JCVinfection, rather than to very low viral levels.

Example 4(c): Assessment of BKV-JCV Cross Reactivity

Assignment of a single conservative cut-point that controls thefalse-negative rate at 0% is unlikely to exclude detection of antibodiesthat cross-react to other common polyoma viruses (false positives), suchas anti-BKV antibodies, which share high identity to JCV in the VP1capsid protein. Additionally, such antibody cross-reactivity may occurthrough exposure of conserved viral epitopes when the HPVLP is directlycoated onto the ELISA plate. Because dual infections with BKV and JCVmay occur in humans and it is not possible to reliably identify patientswho have been infected with BKV and not JCV, the issue ofcross-reactivity was examined in rabbits, a species in which naturalinfection with either BKV or JCV cannot occur.

Rabbits were immunized with BKV by subcutaneous injection of proteins inphosphate-buffered saline without adjuvant, followed by three boosterinjections over a three month period. Serum samples were assayed fordirect binding to JCV or BKV by ELISA. Antisera from BKV-immunizedrabbits bound BKV VLPs with high affinity (EC50=1:100,000) andcross-reacted with HPVLPs with lower affinity (EC50=1:5,000). Pre-immunesera showed no reactivity. Representative data from one rabbit are shownin FIG. 3.

Because BKV antibodies cross-reacted with JCV, thus producing a falsepositive signal in the anti-JCV assay (FIG. 3), low level reactivityagainst JCV in humans could represent low affinity anti-BKV antibodiesthat cross-react with JCV to produce false-positive signals.

Example 4(d): Measuring JCV-Specific Antibody Response (SupplementalConfirmation ELISA)

To distinguish patients with JCV-specific antibodies from those withpotentially low affinity, cross reactive antibodies, a competition ELISAwas developed using soluble HPVLP (secondary assay). JCV-specific higheraffinity antibodies were expected to be more effectively competed by thesoluble antigen, whereas lower affinity antibodies may detach from thecomplexes formed with the JCV antigen in solution and bind to the JCVVLP coated on the ELISA plate. A subset of 515 serum samples fromuropositive (n=204) and uronegative (n=311) patients was systematicallyand non-proportionally sampled for evaluation in the ELISA afterpre-adsorption with either soluble JCV VLP or assay buffer. In FIGS. 4Aand 4B, the reactivity of serum samples from uronegative or uropositivepatients in the screening and confirmation assays are shown side byside. Samples with strong JCV reactivity were highly inhibited bypre-adsorption of antibodies with soluble JCV, while samples with lowlevels of JCV antibodies showed differential competition. The antibodyresponses in most uropositive patients were strongly competed (FIG. 4B).These results support the idea that a significant proportion of the lowserum reactivity to JCV may be due to cross-reactivity of antibodies notspecific to JCV.

The distribution of the serum responses in the confirmation ELISAconsisted of two defined peaks, most optimally separated at 40%inhibition (FIG. 5A) corresponding approximately to the lower 5thpercentile of the response distribution of uropositive samples (FIG.5B). Therefore, the 40% inhibition level was selected as the cut pointfor the confirmation ELISA.

Example 4(e): Finalized Two-Step Anti-JCV Serological Assay

By combining the screening and confirmation assays, the chance ofdetecting samples with “true” JCV-specific antibodies is greatlyenhanced. In the final analysis, samples with nOD₄₅₀ values <0.10 in thescreening ELISA are considered negative for JCV antibodies, and thosewith nOD₄₅₀ values >0.25 in the screening ELISA are considered positivefor JCV antibodies. Samples with reactivity between nOD values 0.10 to0.25 were further tested in the confirmation ELISA. In the confirmationELISA, all samples exhibiting >40% inhibition are classified as positive(FIG. 4). At nOD₄₅₀ values >0.25 the probability of observing >40%inhibition was approximately 95%.

Example 4(f): JCV Seropositivity in the STRATA Cohort and False-NegativeRate

Based on the above algorithm, the seroprevalence rate in STRATApopulation was estimated as 53.6% with bootstrap determined 95%confidence limits ranging from 49.9% to 57.3% [0.536=0.451 (probabilityof the screening ELISA nOD₄₅₀>0.25)+0.085 (probability of screeningELISA nOD₄₅₀ falling between 0.10 and 0.25, and the supplementalconfirmation ELISA %-inhibition >40%)]. This seroprevalence calculationassumed confirmation of anti-JCV antibodies in equal proportions ofsamples from uropositive and uronegative subjects in the nOD regionbetween 0.10 and 0.25. (percent inhibition >40%); this assumption wassupported by a 2-sided Fisher's exact test with a p-value of 0.702.

Of the 204 uropositive patients, five had nOD₄₅₀ between 0.10 and 0.25and did not confirm as having anti-JCV specific antibodies (percentinhibition ≤40%; FIG. 4B).

Example 5: Assay Validation

Assay validation was performed by Focus Diagnostics, Inc. (Cypress, CA),where performance parameters including inter- and intra-assay precision,specificity, sensitivity and stability of assay reagents and controlswere demonstrated. Assay performance parameters including inter- andintra-assay precision, specificity, sensitivity and stability of assayreagents and controls was demonstrated. Precision parameters wereevaluated by three independent analysts in both plasma and serum on fourdifferent days using independent preparations of assay controls. Fordemonstration of assay specificity, ten individual serum and plasmasamples from healthy volunteers or MS patients (TYSABRI® (natalizumab)naïve) were pre-incubated with either assay buffer or a definedconcentration of HPVLP or BKV VLP in solution. Robustness was evaluatedby varying the upper and lower limits of incubation times for sample,conjugate, and substrate addition steps and different lots of HPVLPcoating reagent were evaluated to demonstrate consistent assay controlperformance. Matrix interference was evaluated by determining percentrecovery in samples spiked with pre-defined concentrations of anti-JCVantibodies and by spiking samples containing JCV-specific antibodieswith varying concentrations of irrelevant human monoclonal antibodies.

Example 6: Determination of JCV Antibody Status in PML Patients

Plasma and serum samples (single time-points randomly selected fromserial collections) were obtained from a total of 831 patients from theSafety of TYSABRI® Re-dosing And Treatment (STRATA) study. STRATA is anopen-label, single-arm, multinational study (North America, Europe,Australia, and New Zealand) in which all patients receive natalizumab300 mg by intravenous infusion every 4 weeks for 48 weeks. Urine samplescollected according to the STRATA protocol were analyzed for thepresence of JCV DNA.

From the marketing approval of TYSABRI® in June 2006 to Feb. 9, 2010,there were 35 reported cases of PML on natalizumab treatment. Inaddition, there were three PML cases in the pre-approval clinical trialsof natalizumab (10, 13, 25). Stored samples were obtained from as manyPML cases as possible from time points prior to PML diagnosis (pre-PML).Plasma or serum samples were only available from 11 natalizumab-treatedPML patients (10 MS patients and 1 Crohn's patient: Table 1). Serumsamples were tested that were obtained one to three years prior to PMLdiagnosis. Nearly all of these samples had been collected from patientsparticipating in registries or clinical studies and were stored at -70°C. until analysis. Notably, anti-JCV antibodies were detected in all 11patients (100%) via the combination of the serological status screeningELISA and the supplemental confirmation ELISA (FIGS. 6A and 6B)described above. Using a one-sample Fisher's exact test, this result wassignificantly different from the expected proportion (53.6%) with ap-value of 0.002.

These data indicate that the assay of the present invention can be usedto determine the presence or absence of JCV antibody in subjects as partof an overall evaluation of risk for contracting PML.

TABLE 1 Samples from 11 natalizumab-treated PML patients who hadavailable blood samples prior to diagnosis. PML Natalizumab ExposureDiagnosis No. of doses Final Immunosuppressant Use Subject SourceGeography (date) or months dose Type Duration 1 Clinical Study* BelgiumMarch 5 June Infliximab 32 months 2005 doses 2003 Azathioprine 73 months2 Clinical Study United States Febuary 28 December None (SENTINEL) 2005doses 2004 3 Clinical Study United States Febuary 37 January None(SENTINEL) 2005 doses 2005 4 Post-Marketing Sweden July 17 June None2008 months 2008 5 Clinical Study Germany June 34 doses AprilMitoxantrone 11 months (STRATA) 2009 2009 6 Clinical Study France June35 May Mitoxantrone 10 months (STRATA) 2009 doses 2009 7 Post-MarketingSweden June 29 June None 2009 months 2009 8 Post-Marketing SwitzerlandAugust 28 doses/ June Mitoxantrone 18 months 2009 25 months 2009Azathioprine 21 months 9 Post-Marketing Switzerland October 36 SeptemberMitoxantrone 4 years 2009 months 2009 10 Clinical Study Czech RepublicOctober 44 September Azathioprine  3 months (STRATA) 2009 doses 2009 11Post-Marketing United States October 33 September Methotrexate Unknown2009 doses 2009 *Crohn's Disease; SENTINEL = Safety and Efficacy ofNatalizumab in Combination with Interferon Beta-1a in Patients withRelapsing Remitting Multiple Sclerosis; STRATA = Safety of TYSABRIRe-dosing and Treatment; qd = 4 × day; qwk = 1 × week SENTINEL = Safetyand Efficacy of Natalizumab in Combination with Interferon Beta-1a inPatients with Relapsing Remitting Multiple Sclerosis; STRATA = Safety ofTYSABRI ® Re-dosing and Treatment; ROW = Rest of World; qd = 4 × day;qwk = 1 × week; *Both prior and concurrent treatment with natalizumab

Longitudinal data from other subjects taking an immunomodulator werealso evaluated (i.e., multiple samples collected at different times froma single individual). The longitudinal data indicated that, unliketesting intermittent urinary DNA shedding, the HPVLP assay can reliablybe used to evaluate anti-JCV antibody status, and that JCV antibodystatus remains relatively stable (in the absence of de novo infection).

Example 7: Alternate JCV VP1-VLP Purification Method

This method is an example of an alternative to thedensity-gradient/ultracentrifugation method described above for thepurification of JCV VP1-VLP's from insect cells. The general steps inthe protocol are lysis, BENZONASE® treatment, deoxycholateprecipitation, ammonium sulfate precipitation andconcentration/diafiltration, with a final ion-exchange step using TMAEfractogel.

Sf9 cells infected with JCV-VP1 baculovirus were lysed in PBS, 0.1 mMCaCl₂) by passing twice through a microfluidizer cell disrupter at 5,000psi. Cell debris was removed by low speed centrifugation and thesupernatant treated with 40 units/ml BENZONASE® (EMD Biosciences71206-3) for 1 hour at room temperature. For the deoxycholateprecipitation step, one tenth volume 2.5% deoxycholate was added to thelysate (0.25% final deoxycholate), and the lysate was incubated at 37°C. for 1 hour with gentle stirring. An equal volume of 4 M NaCl, 0.1 mMNaCl was added to the lysate and the lysate was incubated on ice for 1hour. Precipitate was removed by low speed centrifugation. Thesupernatant was then precipitated with 40% ammonium sulfate to removecontaminating proteins. The final 40% was achieved by using 232 g solidammonium sulfate per liter of solution. While mixing the solution gentlyat 4° C., ammonium sulfate was added one fifth at a time, allowing eachaddition to dissolve for 10 to15 minutes before adding the nextfraction. The solution was stirred gently overnight at 4° C. Theammonium sulfate precipitate was removed by low speed centrifugation andthe VP1-containing supernatant was filtered using a 0.45 μm filter andcarried on to the next step. The solution was concentrated 5 to 10 foldusing a 100 kDa NMWL TFF membrane (PELICON®-2 Mini UF Mod Biomax-100 C0.1 m², P2B100001) and exchanged into assembly buffer (25 mM tris, 150mM NaCl, 1 mM CaCl₂), pH 7.5) by diluting 5 fold and concentrating backto the starting volume twice. The solution was stored at 4° C. for >/=36hours. The solution was then diafiltered using a 500 kDa NMWL TFFmembrane (PELICON®-2 Mini UF Mod Biomax-500 V, Millipore part#P2B500V01) using 40 volumes TMA chromatography buffer (25 mM tris, 150mM NaCl, 0.1 mM CaCl₂), pH 8.0). For the chromatography, approximately 1ml resin is required per 2 g starting cell mass. The protein was loadedonto the appropriately sized TMAE column (Fractogel® EMD TMAE HiCap (M)EMD Biosciences cat. 1.10316) and washed with 3 column volumeschromatography buffer. The VLPs were eluted with 25 mM tris, 600 mMNaCl, 0.1 mM CaCl₂), pH 8.0. VP1 purity was assessed by SDS-PAGE andmass spectrometry, presence of VLPs was confirmed by electronmicroscopy, and the percentage of total protein in the form of VLPs wasdetermined by sedimentation velocity analytical ultracentrifugation.This method resulted in HPVLP preparations of about 80% HPVLPs.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims. PML diagnosis (pre-PML). Plasma or serum samples were onlyavailable from 11 natalizumab-treated PML patients (10 MS patients and 1Crohn's patient: Table 1). Serum samples were tested that were obtainedone to three years prior to PML diagnosis. Nearly, all of these sampleshad been collected from patients participating in registries or clinicalstudies and were stored at −70° C. until analysis. Notably, anti-JCVantibodies were detected in all 11 patients (100%) via the combinationof the serological status screening ELISA and the supplementalconfirmation (FIGS. 6A and 6B) described above. Using a one-sampleFisher's exact test, this result was significantly different from theexpected proportion (53.6%) with a p-value of 0.002.

These data indicate that the assay of the present invention can be usedto determine the presence or absence of JCV antibody in subjects as partof an overall evaluation of risk for contracting PML.

What is claimed is:
 1. A reaction mixture comprising soluble JC Virus(JCV) virus-like particles (VLP), a sample of serum or plasma from asubject, and immobilized JCV VLP, wherein the immobilized JCV VLP hasbeen coated with a blocking agent.
 2. The reaction mixture of claim 1,wherein the soluble JCV VLP are highly purified virus like particles(HPVLP).
 3. The reaction mixture of claim 2, wherein at least 20% of theHPVLPs comprise more than five VP1 polypeptides in an HPVLP.
 4. Thereaction mixture of claim 2, wherein at least 70% of the HPVLPs comprisemore than five VP1 polypeptides in an HPVLP.
 5. The reaction mixture ofclaim 2, wherein an HPVLP consists essentially of VP1 polypeptides. 6.The reaction mixture of claim 2, wherein an HPVLP further comprises atleast one of a VP2, or a VP3.
 7. The reaction mixture of claim 2,wherein the VP1 in the HPVLP is a recombinant VP1.
 8. The reactionmixture of claim 2, wherein at least one VP1 in the HPVLP is a mutantVP1.
 9. The reaction mixture of claim 1, wherein the immobilized JCV VLPare HPVLP.
 10. The reaction mixture of claim 9, wherein wherein at least20% of the HPVLPs comprise more than five VP1 polypeptides in an HPVLP.11. The reaction mixture of claim 9, wherein at least 70% of the HPVLPscomprise more than five VP1 polypeptides in an HPVLP.
 12. The reactionmixture of claim 9, wherein an HPVLP consists essentially of VP1polypeptides.
 13. The reaction mixture of claim 9, wherein an HPVLPfurther comprises at least one of a VP2, or a VP3.
 14. The reactionmixture of claim 9, wherein the VP1 in the HPVLP is a recombinant VP1.15. The reaction mixture of claim 9, wherein at least one VP1 in theHPVLP is a mutant VP1.
 16. The reaction mixture of claim 1, wherein thesample is from a subject prescribed an immunomodulator, a subjectconsidering taking an immunomodulator, or a subject suspected of havingProgressive Multifocal Leukoencephalopathy (PML).
 17. The reactionmixture of claim 1, wherein the blocking agent is a nonspecific proteinthat is antigenically neutral.
 18. The reaction mixture of claim 17,wherein the blocking agent is selected from bovine serum albumin (BSA),casein, or a solution of milk powder.
 19. The reaction mixture of claim1, wherein the reaction mixture comprises anti-JCV serum antibodiesbound to the soluble JCV VLP.
 20. The reaction mixture of claim 19,wherein soluble JCV VLP and anti-JCV serum antibodies in the reactionmixture comprise a pre-formed complex.
 21. A method comprising: (a)forming a reaction mixture comprising soluble JC Virus (JCV) VLP and analiquot of a sample of serum or plasma from a subject, therebyperforming a pre-treatment reaction mixture; and (b) incubating saidreaction mixture of (a) under conditions suitable for the solubleJCV-VLP to bind anti-JCV antibodies in the reaction mixture, if present,thereby performing a pre-treatment step.
 22. The method of claim 21,wherein the method further comprises: (c) after the pre-treatment,contacting the incubated pre-treatment reaction mixture with immobilizedJCV VLP under conditions suitable for antibodies against other polyomaviruses that cross-react with JCV VLP to bind the immobilized JCV VLP,if present, and wherein the immobilized JCV VLP has been coated with ablocking agent.
 23. The method of claim 22, wherein the anti-JCVantibodies in the sample, if present, are more effectively competed bythe soluble JCV VLP, whereas the antibodies against other polyomaviruses that cross-react with JCV VLP, if present, preferentially detachfrom the complexes formed with the JCV VLP in solution and bind to theimmobilized JCV VLP.
 24. The method of claim 23, wherein the methodcomprises detecting antibodies from the sample aliquot that are bound tothe immobilized JCV VLP, thereby performing a competition assay.
 25. Themethod of claim 24, wherein the method comprises: (d) forming a reactionmixture comprising a second aliquot of the sample, wherein the reactionmixture comprises substantially no soluble JCV VLP; (e) contacting thereaction mixture of (d) to a substrate comprising immobilized JCV underconditions suitable for anti-JCV antibodies in the second aliquot tobind the immobilized JCV, if present; and (f) detecting antibodies fromthe second aliquot that are bound to the immobilized JCV VLP, ifpresent, thereby performing a primary assay.
 26. The method of claim 25,wherein the method further comprises: (g) comparing a detected level ofantibodies that are bound to the immobilized JCV VLP in the competitionassay to a detected level of antibodies that are bound to theimmobilized JCV VLP in the primary assay, such that a decrease in thedetected level in the competition assay compared to the primary assayindicates the sample is positive for anti-JCV antibody, and a change inthe detected level below a specified percentage indicates that there isno JCV-specific antibody present in the sample.